Abstract Exposure of humans and laboratory animals to acrylamide (ACR) produces cumulative neurotoxicity characterized by gait abnormalities, muscle weakness and a central-peripheral neuropathy. ACR is an 1,2-unsaturated carbonyl derivative and is classified as a type-2 alkene. This is a large class of electrophilic chemicals that have broad industrial, agricultural and pharmaceutical uses. These chemicals are also well-recognized dietary contaminants and environmental pollutants. Data collected during yrs. 17-20 have provided evidence that ACR impairs nerve terminal function by forming irreversible covalent adducts with nucleophilic sulfhydryl groups on functionally important proteins. Proteomic analyses indicate that the protein targets of ACR and the type-2 alkenes are also acceptors for nitric oxide (NO) signaling. NO is a biological electrophile and has been classically thought to influence cell processes through guanylyl cyclase activation. However, NO can also modulate cell physiology by forming reversible adducts with cysteine thiolates in protein catalytic triads. At the nerve terminal, NO signaling is critically involved in neurotransmission through modulation of the synaptic vesicle cycle and other presynaptic processes. Thus, NO and ACR interact at common cysteine sulfhydryl sites and, therefore, we hypothesize that irreversible adduction of these receptors by ACR blocks reversible NO binding. The disruption of NO signaling and ensuing loss of neuromodulatory control produces presynaptic toxicity. Therefore, Specific Aim #1 research will define the interactions of ACR with the S-nitrosylated (SNO) proteome of CNS nerve terminals. SNOSID (S-nitrosylated site identification) proteomic analysis will be used to demonstrate ACR adduction of SNO-cysteine sites on nerve terminal proteins. Specific Aim #2 studies will evaluate the specificity of the ACR-NO interaction by considering alternative mechanisms of action;i.e., we will determine the effects of ACR on soluble quanylyl cyclase and nitric oxide synthase (NOS) activity/gene expression. Because NO modulates physiological processes in most cells, it is unclear why nerve terminal NO signaling might be selectively targeted by ACR. Therefore, Specific Aim #3 studies will consider several anatomical and molecular features that might predispose nerve terminals to electrophilic attack. Identifying the mechanism of ACR neurotoxicity could offer global insight regarding the toxicological processes of other type-2 alkenes. Results of the proposed research could also help us understand the pathogenesis of Alzheimer's disease (AD) and other chronic neurodegenerative conditions that presumably involve cellular oxidative stress and endogenous generation of acrolein and other type-2 alkenes. PUBLIC HEALTH RELEVANCE Human exposure to conjugated type-2 alkenes (e.g., acrylamide, methyl acrylate, methylvinyl ketone) occurs through pervasive environmental sources (e.g., industrial exposure, cigarette smoking, car exhaust, combustion, pharmaceuticals) and can result in significant toxicity in nervous tissue and other organ systems (liver, kidney). There is also evidence that endogenous production of type-2 alkenes (e.g., acrolein, 2-hydryoxy-4-nonenal) is critically involved in mediating nerve cell injury associated with accidental neurotrauma and certain human neurodegenerative conditions such as Alzheimer's disease. Therefore, the proposed studies of type-2 alkene neurotoxicity could lead to a better understanding of brain injuries caused by environmental toxicant exposure or disease processes, which would ultimately help in the development of effective therapeutic approaches.